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Enzymatic assembly of overlapping DNA molecules and then incubated at 50 1C for as few
as 15 min (Online Methods). This approach dramatically simplifies
DNA molecules up to the construction of large DNA molecules from constituent parts.
Exonucleases that recess double-stranded DNA from 5 ends will
several hundred kilobases not compete with polymerase activity. Thus, all enzymes required
for DNA assembly can be simultaneously active in a single
isothermal reaction. Furthermore, circular products can be
Daniel G Gibson1, Lei Young1, Ray-Yuan Chuang1, enriched as they are not processed by any of the three enzymes in
J Craig Venter1,2, Clyde A Hutchison III2 & the reaction. We optimized a 50 1C isothermal assembly system
Hamilton O Smith2 using the activities of the 5 T5 exonuclease (Epicentre), Phusion
DNA polymerase (New England Biolabs (NEB)) and Taq DNA
2009 Nature America, Inc. All rights reserved.
here), individual reactions were carried out in only two steps. Here Anneal at 50 C Phusion polymerase
we improved this two-step thermocycled method by using exo- T5 exonuclease Taq ligase
5 3 5 3
3 5 3 5
nuclease III and antibody-bound Taq DNA polymerase, which
Repair at 50 C with Phusion polymerase and Taq ligase
allow for one-step thermocycled in vitro recombination (Supple-
mentary Fig. 4 and Supplementary Results online). 3
5
5
3
1The J. Craig Venter Institute, Synthetic Biology Group, Rockville, Maryland, USA, and 2San Diego, California, USA. Correspondence should be addressed to
D.G.G. (dgibson@jcvi.org).
RECEIVED 5 JANUARY; ACCEPTED 16 MARCH; PUBLISHED ONLINE 12 APRIL 2009; DOI:10.1038/NMETH.1318
16
6 (iii)
0
(kb) M
8
10
20
overlapping by B450 bp at the termini (black,
0.
0.
4
0
4 (i) (iv) (kb) 40
overlaps labeled A and B), were reacted for 3 (ii) 6
A Not l
A (i) B 4 20
016 min to form a 6-kb circle, pRS415 (iv). Linear B 3 8 Input
B
assembly products (iii) were then removed by (iii)
5 DNA
A A
incubation with additional tenfold excess T5 A (ii) B A B
B
exonuclease after the 16-min incubation (T5). (iv)
48.5
(f) Not I digestion of BACs purified from ten
E. coli clones after electroporation of the assembly
reaction shown in e. The correct insert size (310 kb) is indicated by the arrow. (g) Assembly of quarter M. genitalium genomes C124, C2549, C5077 and
C78101 (ref. 11) to produce a complete M. genitalium genome. DNA products were analyzed by conventional gel electrophoresis (a,b) and by field-inversion gel
electrophoresis (cg). M, 1-kb DNA extension markers; l, lambda markers.
6-kb linear fragment (Fig. 2b). We concluded that DNA molecules molecules (210 repaired junctions) after two-step thermocycled
can be assembled and repaired in a single isothermal step using assembly revealed only 4 errors (Supplementary Table 1 online).
this method. This equates to only about 1 error per 50 DNA molecules joined.
We next determined whether DNA molecules with overlaps of Therefore, if our hierarchical scheme to assemble the M. genitalium
only 40 bp could be joined. We accomplished this when we reduced genome was used11 without sequence verification at intermediate
the concentration of T5 exonuclease (Fig. 2c). Three 5-kb DNA steps, 34 errors would likely be present. We expect that the number
fragments, F1F3, were efficiently assembled into an 8-kb bacterial of mutations would be even lower with the isothermal assembly
artificial chromosome (BAC). Furthermore, when we transformed system because gaps are filled in by Phusion DNA polymerase,
these assembled DNA molecules into E. coli, we obtained 4,500 which has higher fidelity than Taq polymerase.
colonies, and nine out of ten colonies tested had the predicted Our isothermal method can be used to assemble DNA molecules
15-kb insert (Fig. 2d). of unprecedented sizes, and we used it to assemble the complete
During the construction of the synthetic M. genitalium genome, synthetic 583-kb M. genitalium genome (Fig. 2g). The size limit for
we could not use our two-step thermocycled in vitro recombination in vitro DNA assembly is not known, but products as large as 900 kb
method to clone assembled DNA molecules larger than B150 kb in have been observed (Supplementary Fig. 6 online). Of the three
E. coli11. To determine whether the isothermal assembly method in vitro recombination methods, we prefer the one-step-isothermal
could be used to join and clone DNA fragments of larger size, system because of its simplicity. This approach could be very useful
we reacted two synthetic M. genitalium quarter DNA molecules, for cloning multiple inserts into a vector without relying on the
C2549 (144 kb) and C5077 (166 kb), with BAC2577 (8 kb), a availability of restriction sites and for rapidly constructing large
cloning vector specific for the assembly of these two DNA mole- DNA molecules. For example, regions of DNA too large to be
cules. The 318-kb Mgen2577 product was efficiently produced, so amplified by PCR can be divided into multiple overlapping PCR
we conclude that DNA fragments this size can be joined by this amplicons and then assembled into one piece. The one-step
method (Fig. 2e). To determine whether this method could be used thermocycled method could be used to generate linear assemblies
to clone assembled DNA fragments this size, we transformed a as the exonuclease is inactivated during the reaction (Supplemen-
fraction of this assembly reaction into E. coli. We obtained several tary Figs. 4 and 5).
hundred clones, and 5 out of 10 colonies screened had the correct Synthetic biologists are engineering genetic pathways for the
insert size (310 kb; Fig. 2f). Thus, this DNA assembly system can be production of biofuels, pharmaceuticals and industrial com-
used to join and clone DNA molecules up to several hundred pounds14,15. Here we provide efficient methods for constructing
kilobases in length in E. coli, the approximate upper limit for these pathways, from natural or synthetic DNA.
transformation into this bacterium13. In a direct comparison of all
our assembly methods, we found that only the one-step in vitro METHODS
recombination methods could be used to clone assembled DNA Methods and any associated references are available in the online
fragments this size (Supplementary Fig. 5 online). version of the paper at http://www.nature.com/naturemethods/.
During in vitro recombination, errors may be introduced in
the assembled DNA. However, sequencing of 30 cloned DNA Note: Supplementary information is available on the Nature Methods website.
ACKNOWLEDGMENTS 2. Smith, H.O. & Wilcox, K.W. J. Mol. Biol. 51, 379391 (1970).
This work was supported by the Office of Science (Biological and Environmental 3. Shetty, R.P., Endy, D. & Knight, T.F. Jr. J. Biol. Eng. 2, 5 (2008).
Research) United States Department of Energy grant number DE-FG02-02ER63453, 4. Yount, B., Denison, M.R., Weiss, S.R. & Baric, R.S. J. Virol. 76, 1106511078
and Synthetic Genomics, Inc. (2002).
5. Horton, R.M., Cai, Z.L., Ho, S.N. & Pease, L.R. Biotechniques 8, 528535
AUTHOR CONTRIBUTIONS (1990).
D.G.G., L.Y., R.-Y.C., J.C.V., C.A.H. and H.O.S. designed research; D.G.G., L.Y., 6. Horton, R.M. Mol. Biotechnol. 3, 9399 (1995).
R.-Y.C., C.A.H. and H.O.S. performed research; D.G.G., L.Y., R.-Y.C., J.C.V., C.A.H. 7. Bang, D. & Church, G.M. Nat. Methods 5, 3739 (2008).
and H.O.S. analyzed data; and D.G.G., C.A.H. and H.O.S. wrote the paper. 8. Geu-Flores, F., Nour-Eldin, H.H., Nielsen, M.T. & Halkier, B.A. Nucleic Acids Res.
35, e55 (2007).
COMPETING INTERESTS STATEMENT
9. Aslanidis, C. & de Jong, P.J. Nucleic Acids Res. 18, 60696074 (1990).
The authors declare competing financial interests: details accompany the full-text
10. Li, M.Z. & Elledge, S.J. Nat. Methods 4, 251256 (2007).
HTML version of the paper at http://www.nature.com/naturemethods/.
11. Gibson, D.G. et al. Science 319, 12151220 (2008).
12. Sayers, J.R. & Eckstein, F. J. Biol. Chem. 265, 1831118317 (1990).
Published online at http://www.nature.com/naturemethods/
13. Sheng, Y., Mancino, V. & Birren, B. Nucleic Acids Res. 23, 19901996
Reprints and permissions information is available online at (1995).
http://npg.nature.com/reprintsandpermissions/
14. Endy, D. Nature 438, 449453 (2005).
1. Gellert, M. Proc. Natl. Acad. Sci. USA 57, 148155 (1967). 15. Drubin, D.A., Way, J.C. & Silver, P.A. Genes Dev. 21, 242254 (2007).
2009 Nature America, Inc. All rights reserved.
for 15 to 60 min (60 min was optimal). prepared from these cells by alkaline lysis using the P1, P2 and P3
buffers (Qiagen) followed by isopropanol precipitation. DNA
Rolling-circle amplification (RCA) of assembled products. RCA pellets were dissolved in TE buffer containing RNase and then
was carried out as previously described18. One microliter of the digested with Not I to release the insert from the BAC.
repaired or unrepaired reaction was mixed with 1 ml of 100 mM
NaOH and incubated at room temperature (1822 1C) for 5 min Agarose gel analyses of assembled DNA molecules and cloned
to denature the double-stranded DNA. One microliter of this products. U-5 field-inversion gel electrophoresis analysis was
alkaline-treated mixture was then added to 19 ml of RCA compo- performed on 0.8% E-gels (Invitrogen) and the parameters were
nents in a 0.2 ml PCR tube. The final reaction concentrations for forward 72 V, initial switch 0.1 s, final switch 0.6 s, with linear
RCA are as follows: 37 mM Tris-HCl pH 7.5, 50 mM KCl, 10 mM ramp and reverse 48 V, initial switch 0.1 s, final switch 0.6 s, with
MgCl2, 5 mM (NH4)2SO4, 100 mg ml1 BSA, 1 mM DTT, linear ramp. U-2 field-inversion gel electrophoresis analysis was
3.25 mM random hexamers (Fidelity System), 1 U ml1 yeast performed on 1% agarose gels (BioRad) in 1 TAE buffer with
pyrophosphatase (United States Biochemical) and 250 U ml1 0.5 mg ml1 ethidium bromide without circulation, and the para-
phi29 DNA polymerase (NEB). The reaction was incubated at meters were forward 90 V, initial switch 5.0 s, final switch 30 s, with
30 1C for 20 h then terminated by incubation at 65 1C for 10 min. linear ramp, and reverse 60 V, initial switch 5.0 s, final switch 30 s,
with linear ramp. DNA bands were visualized with a BioRad Gel
Cloning the DNA assembly products. To clone assembled pro- Doc or an Amersham Typhoon 9410 Fluorescence Imager.
ducts, reactions were carried out in the presence of PCR-amplified
16. Lartigue, C., Duret, S., Garnier, M. & Renaudin, J. Plasmid 48, 149159 (2002).
BACs containing 40 bp of overlapping sequence to the ends of the 17. Henikoff, S. Gene 28, 351359 (1984).
assembled product. Not I restriction sites were also included to 18. Hutchison, C.A., III, Smith, H.O., Pfannkoch, C. & Venter, J.C. Proc. Natl. Acad.
allow release of the vector11. To produce BAC-F1/F3, primers Sci. USA 102, 1733217336 (2005).